A method for lipid analysis in biological samples

Abstract

The present invention provides a method for lipid analysis in biological samples. The method of the present application includes processes such as lipid extraction, separation and purification, and high-resolution mass spectrometry detection in biological samples, and specifically includes the following steps: rapid thin-layer chromatography analysis after lipid extraction from biological samples to determine the lipid classes contained; determining the silica gel column separation method and steps in combination with the analysis results of thin-layer chromatography; further separating and purifying the lipids using a silica gel chromatographic column, then eluting and separating through liquid chromatography, and entering high-resolution mass spectrometry detection and analysis. The method of the present application also includes using software to analyze and process the detected lipids, and finally determining the lipid species information. The method of the present invention can significantly improve the lipid purity and the separation degree of its isomers, enhance the low-abundance lipid signals, reduce the lipid co-elution behavior, obtain high-definition secondary mass spectra, effectively improve the accuracy and coverage of lipid qualitative and quantitative analysis, and has simple operation, high repeatability, and strong versatility.

Description

Technical Field

[0001] The present invention belongs to the fields of analytical chemistry and lipidomics research, and particularly relates to a method for lipid analysis in biological samples. Background Art

[0002] Lipid compounds are very important substances in organisms. As the main components of biological membranes, lipid compounds have molecular diversity and various biological functions, including providing a suitable environment for protein interactions, storing energy, and serving as important messenger molecules and other important physiological functions. In recent years, they have received much attention in the research of various diseases and biological problems, such as being closely related to apoptosis, signal transduction, disease infection, immune function, growth and development, food nutrition, etc. At the same time, lipid metabolism is also closely related to various diseases such as diabetes, liver cancer, kidney disease, breast cancer, etc.

[0003] There are many types of lipids with complex and diverse structures, and there are a large number of isomers, including acyl isomers, double bond isomers, etc. At present, the identification of lipids mainly relies on high-quality secondary mass spectrometry diagrams for structure prediction. Therefore, obtaining high-definition and high-quality secondary mass spectrometry diagrams is the key to accurate lipid identification. Ultra-high performance liquid chromatography (high performance liquid chromatography)-high resolution mass spectrometry technology (HPLC-HRMS) realizes the effective separation of lipids through chromatography, and at the same time combines the high selectivity, high sensitivity and qualitative ability of mass spectrometry, achieving the complementary advantages of chromatography and mass spectrometry. It is the most widely used and key application platform for lipidomics research. In the data-dependent acquisition (DDA) mode, after the mass spectrometer performs a full scan, it performs a secondary scan on the list of precursor ions (intensity-dependent) selected in the full scan mass spectrum. It is the most commonly used scan mode for lipid identification currently. Analyzing ion selection depends on data intensity, which is not friendly to the detection of low-abundance and low-response lipids. At the same time, the DDA scan mode usually also needs to set an isolation window of 0.5 to 3 m / z, so that its fragment spectrum is also a convolution fragment peak from multiple precursor ions. The characteristics of lipids determine that when separated by a single chromatographic column, there will inevitably be a large number of co-elution phenomena between and within lipid classes, thus affecting the purity of the data points collected by the mass spectrometer. Although mass spectrometry data can usually be deconvoluted by a mathematical algorithm through data processing software to re-analyze the components not separated by chromatography and restore their true mass spectrometry information. However, deconvolution is based on algorithms and there will still be errors. Therefore, a comprehensive and highly accurate qualitative and quantitative analysis of lipids in biological samples remains a challenge. Summary of the Invention

[0004] The purpose of this application is to provide a general deep lipid detection and analysis method and operation process for biological samples to solve the problems that the quality of the secondary mass spectrometry diagram is not high-definition due to current lipid co-elution, and the response signal of low-abundance lipids is too low to be detected, affecting the comprehensive characterization of lipids in biological samples.

[0005] To achieve this object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for lipid analysis in biological samples. The method for lipid analysis in biological samples comprises the following steps:

[0007] (1) Extract lipids from the biological sample to obtain a first sample;

[0008] (2) Use thin-layer chromatography to determine the lipid classes of the first sample. The lipid classes include non-polar lipids, medium-polar lipids, and polar lipids;

[0009] (3) According to the lipid classes and contents in (2), use a silica gel chromatography column to separate and purify the first sample to obtain a second sample:

[0010] (4) Perform gradient elution on the second sample by liquid chromatography and then conduct mass spectrometry detection and analysis.

[0011] In some embodiments, the silica gel chromatography column in step (3) is an NH2-based SPE column. The eluent of the silica gel chromatography column includes one or more of a n-hexane phase, an isopropanol:dichloromethane (3:7 (v / v)) phase, and a methanol phase; wherein, the n-hexane phase is used to elute non-polar lipids; the isopropanol:dichloromethane (3:7 (v / v)) phase is used to elute medium-polar lipids; and the methanol phase is used to elute polar lipids.

[0012] In some embodiments, the elution volume of each eluent is 3 to 6 times the column volume.

[0013] In some embodiments, the silica gel chromatography column is activated with 6 times the column volume of n-hexane.

[0014] In some embodiments, in step (3), after the first sample is concentrated to dryness, it is redissolved with n-hexane and then loaded onto the column.

[0015] In some embodiments, step (3) includes concentrating the first sample to dryness, redissolving it with n-hexane and then loading it onto the column; eluting weakly polar lipids with n-hexane in sequence, eluting medium-polar lipids with isopropanol:dichloromethane (3:7 (v / v)), and eluting strongly polar lipids with methanol. The elution volume is 6 - 12 times the column volume.

[0016] In some embodiments, after the eluate obtained by separating and purifying the first sample with the silica gel chromatography column in step (3) is concentrated to dryness, the sample eluted with the methanol phase is redissolved with pure methanol, and the sample eluted with the n-hexane phase or the isopropanol:dichloromethane (3:7 (v / v)) phase is redissolved with dichloromethane:methanol (1:1 (v / v)), and then filtered through a membrane; as a preferred embodiment, the membrane is a 0.22-micron membrane.

[0017] In some embodiments, the second sample includes one or more of a n - hexane phase elution sample, an isopropanol:dichloromethane (3:7 (v / v)) phase elution sample, and a methanol phase elution sample; in step (4), the chromatographic columns used in the liquid chromatography include one or more of a C30 chromatographic column, a C18 chromatographic column, and a HILIC chromatographic column, wherein the C30 chromatographic column is used for separating and purifying the n - hexane phase elution sample, the C18 chromatographic column is used for separating and purifying the isopropanol:dichloromethane (3:7 (v / v)) phase elution sample, and the HILIC chromatographic column is used for separating and purifying the methanol phase elution sample.

[0018] In some embodiments, the mobile phase system of the C30 chromatographic column or the C18 chromatographic column is:

[0019] A1: acetonitrile: water (6:4 (v / v));

[0020] B1: isopropanol: acetonitrile (9:1 (v / v)), and both A1 and B1 are added with 5 - 10 mM ammonium formate and 0.1% - 0.2% (v / v) formic acid;

[0021] The mobile phase system of the HILIC column is:

[0022] A2: acetonitrile: water (1:3 (v / v));

[0023] B2: acetonitrile: water (95:5 (v / v)), and both A2 and B2 are added with 5 - 10 mM ammonium formate and 0.1% - 0.2% (v / v) formic acid.

[0024] In the present invention, the elution gradient of the chromatography can be adjusted according to the lipids to be analyzed to obtain a good chromatographic separation effect.

[0025] In some embodiments, the liquid chromatography elution procedure in step (4) is:

[0026] C30 chromatographic column elution procedure: at 0 min, mobile phase B1 is 50% v / v; at 4 min, mobile phase B1 is 70% v / v; at 13 min, mobile phase B1 is 75% v / v; at 24 min, mobile phase B1 is 85% v / v; at 33 min, mobile phase B1 is 90% v / v; at 36 min, mobile phase B1 is 92% v / v; at 37 min, mobile phase B1 is 95% v / v; at 46 min, mobile phase B1 is 95% v / v; at 46.1 min, mobile phase B1 is 50% v / v; at 50 min, mobile phase B1 is 50% v / v;

[0027] C18 Column Elution Program: At 0 min, mobile phase B1 is 40% v / v; at 3 min, mobile phase B1 is 50% v / v; at 6 min, mobile phase B1 is 62% v / v; at 14 min, mobile phase B1 is 72% v / v; at 15 min, mobile phase B1 is 85% v / v; at 16 min, mobile phase B1 is 95% v / v; at 19 min, mobile phase B1 is 95% v / v; at 19.1 min, mobile phase B1 is 40% v / v; at 22 min, mobile phase B1 is 40% v / v;

[0028] HILIC Column Elution Program: At 0 min, mobile phase B2 is 99% v / v; at 6 min, mobile phase B2 is 94% v / v; at 11 min, mobile phase B2 is 92% v / v; at 16 min, mobile phase B2 is 88% v / v; at 15 min, mobile phase B2 is 85% v / v; at 16 min, mobile phase B2 is 95% v / v; at 19 min, mobile phase B2 is 95% v / v; at 19.1 min, mobile phase B2 is 40% v / v; at 22 min, mobile phase B2 is 40% v / v.

[0029] In some embodiments, the length of the chromatographic column used in the liquid chromatography is 100 - 150 mm; the injection volume is 2 - 10 μL, the column temperature is 30 - 50 °C, and the flow rate is 0.2 - 0.4 mL / min.

[0030] In some embodiments, in step (4), after the mass spectrometry is collected in the CID mode, the accurate ion mass-to-charge ratio, retention time, and high-definition secondary mass spectrometry diagram of the lipid primary mass spectrometry are obtained.

[0031] In some embodiments, in step (4), the collection method of the mass spectrometry is as follows: the effluent after separation and purification by a C30 chromatographic column is detected and analyzed in the positive ion mode; the effluent after separation and purification by a C18 chromatographic column is detected and analyzed in both positive and negative modes; the effluent after separation and purification by a HILIC chromatographic column is detected and analyzed in both positive and negative modes.

[0032] In some embodiments, the mass spectrometry instruments include Thermo Scientific Q Exactive, Waters, or Agilent.

[0033] In some embodiments, the primary scan range of the mass spectrometry is 50 - 1200 m / z, and the secondary acquisition mass range is 50 - 1200 m / z.

[0034] In some embodiments, the mass spectrometry parameters of the Thermo Scientific Q Exactive are as follows: heated electrospray ionization source, a. the voltage in both positive and negative ion modes is 3.0 - 4.0 kV; b. the temperature of the ion transfer tube is 300 - 320 °C; c. the sheath gas pressure is 30 - 40 psi respectively, and the auxiliary gas heating temperature is 300 °C; d. the scan mode is Full MS, and the NCE is 25 - 50 eV.

[0035] In some embodiments, the mass spectrometry parameters of Waters are as follows: the scan time is 0.5 - 1 s; the collision energy in the high-energy channel is 20 - 50 eV; ESI source, the capillary voltage is 2.0 - 3.0 kv; the ion source temperature is 90 - 110 °C; the cone voltage is 35 - 45 V.

[0036] In some embodiments, the mass spectrometry parameters of Agilent are as follows: the collision energy is 20 - 30 eV, the gas temperature is 250 °C, the sheath gas temperature is 300 °C; the sheath gas flow rate is 12 L / min; the capillary voltage in the positive ion mode is 2500 - 3500 V; the cone voltage is 60 - 65 V; the minimum acquisition rate of MS and MS / MS is 2 - 4 spectra / s.

[0037] In some embodiments, the mass spectrometry parameters of the Thermo Scientific Q Exactive are as follows: heated electrospray ionization source, and the positive and negative modes are used for acquisition respectively; a. the voltage in both positive and negative ion modes is 4.00 KV; b. the temperature of the ion transfer tube is 320 °C; c. the sheath gas pressure is 40 psi and 30 psi respectively, the auxiliary gas pressure is 10 arb, and the auxiliary gas heating temperature is 300 °C; d. the scan mode is Full MS, the instrument resolution is set to 70000, -dd / MS2, the resolution is 17500, the NCE is 30 eV, and the scan range is 50 - 1200 m / z.

[0038] In some embodiments, the mass spectrometry parameters of Waters are as follows: the mass range is 50 - 1200 m / z, the scan time is 0.8 s; the collision energy in the high-energy channel is 20 - 50 eV; ESI source, positive (+) ion mode; the capillary voltage is 2.5 kv(+); negative (-) ion mode; the capillary voltage is 2.0 kv(-); the ion source temperature is 100 °C; the cone voltage is 40 V.

[0039] In some embodiments, the mass spectrometry parameters of Agilent are as follows: electrospray ionization source, data-dependent mode for data acquisition; the mass range for the first-stage mass spectrometry is m / z 100 - 1600, and the mass range for the second-stage mass spectrometry is m / z 50 - 1200; the collision energy in the positive ion mode is 20 eV; the collision energy in the negative ion mode is 25 eV; the gas temperature is 250 °C, and the sheath gas temperature is 300 °C; the sheath gas flow rate is 12 L / min; the capillary voltage in the positive ion mode is 3000 V and in the negative ion mode is 3000 V; the fragmentation voltage is 150 V; the cone voltage is 65 V; the minimum acquisition rate for MS and MS / MS is 3 spectra / s.

[0040] In some embodiments, the column temperature of the chromatographic column is 35 - 50 °C.

[0041] In some embodiments, the injection volume for each injection of the HPLC chromatographic column is 2 - 10 μL.

[0042] In some embodiments, the biological sample includes a sample containing lipids or lipid analogs.

[0043] In some embodiments, the biological sample includes any one of traditional Chinese medicine, biological tissue, food, in vitro detection samples of the human or animal body, and artificial synthetic biological tissue.

[0044] In a specific embodiment of the present invention, the biological sample is milk or zebrafish brain tissue.

[0045] In the method of the present invention, the Folch method is preferably used to extract lipids from biological samples, and other lipid extraction methods are also acceptable.

[0046] In some embodiments, the method for extracting lipids from the biological sample in step (1) is as follows:

[0047] An extraction agent is added to the biological sample, and it is ultrasonically disrupted or allowed to stand. Then, water is added and centrifuged, and the organic phase is collected; the remaining part is extracted again, the organic phases are combined, the solvent is removed, redissolved, and filtered to obtain the first sample; the extraction agent includes methanol and dichloromethane.

[0048] In some embodiments, ultrasonic disruption is performed using an ultrasonic disruptor. In some embodiments, the parameters of the ultrasonic disruptor are: each disruption is for 10 - 20 s, the interval between each disruption is 20 - 40 s, the power is 500 - 650 w, and the total disruption time is 10 - 30 min.

[0049] In some embodiments, the method for removing the solvent is nitrogen blowing.

[0050] In some embodiments, redissolution is performed using methanol and dichloromethane.

[0051] In some embodiments, the volume ratio of methanol to dichloromethane in the extractant is 1:1 - 1:2.

[0052] In some embodiments, the biological sample includes a solid sample and a liquid sample. As a preferred embodiment, the solid sample is ≥20 mg and the liquid sample is ≥100 μL.

[0053] In some embodiments, step (2) includes:

[0054] Spot the first sample and the standard on a silica gel thin-layer chromatography plate;

[0055] Develop by the two-time development method. The first developer includes n-hexane and ether, and the second developer includes dichloromethane, isopropanol, methanol, and formic acid. After development, take it out and wait for the reagent to volatilize completely, then develop it in a color development cylinder. Preferably, the color developer for the two-time development method is iodine.

[0056] In some embodiments, the method of spotting is as follows: Spot the first sample and the standard on a silica gel plate pre-dried at 60 °C for 5 min using a silica gel thin-layer chromatography plate: the spotting diameter is ≤3 mm, 10 mm from the lower edge of the plate, the spotting amount of the standard is 10 μL, and the spotting amount of the first sample is 3 μL.

[0057] In some embodiments, the first developer is a mixed solution of n-hexane and ether, and the second developer is a mixed solution of dichloromethane, isopropanol, methanol, and formic acid.

[0058] In some embodiments, the volume ratio of n-hexane to ether in the first developer is 1:1.

[0059] In some embodiments, the volume ratio of dichloromethane, isopropanol, methanol, and formic acid in the second developer is 5:3:2:0.5.

[0060] In some embodiments, the two-time development method includes developing in the first developer to 45% of the height of the silica gel plate, taking out the silica gel plate from the development cylinder, waiting for the organic solvent to volatilize completely, and then developing in the second developer to 90% of the height of the silica gel plate.

[0061] According to the thin-layer chromatography results in step (2) of the present invention, by comparing the retention factor of the lipid standard on the thin-layer plate and the color and depth of the developed spots, the lipids contained in the sample and their contents can be quickly determined, and the lipid separation and purification method and process are optimized.

[0062] According to the moving distance of lipids in the thin-layer plate, the present invention can divide lipids into non-polar lipids such as triglycerides; medium-polar lipids such as sphingolipids, diglycerides, and sterol lipids; and polar lipids such as phospholipids.

[0063] The present invention separates sample lipids into three parts: non-polar lipids, medium-polar lipids and polar lipids, and further optimizes lipid separation by screening liquid chromatography columns and optimizing the elution gradient of the chromatography columns.

[0064] In some embodiments, the method for analyzing lipids in a biological sample further comprises step (5): analyzing mass spectrometry data.

[0065] In some embodiments, the step (5) includes: using software to deconvolute the mass spectrometry data, matching the lipid database for processing and analysis, preliminarily selecting possible lipid categories, types, and fatty acid chain compositions, and then manually annotating and screening to determine the structure of the isomers and exclude false positives to determine the lipid molecular structure.

[0066] In some embodiments, step (5) includes: processing data using MSDIAL, selecting identified lipids according to the highest score, selecting lipids with fill%>50 in QC samples (quality control samples), RSD<30%, and analyzing after deleting duplicates.

[0067] Beneficial effects:

[0068] In view of the currently commonly used lipid analysis methods, this application optimizes the pre-treatment to elute lipids according to different polarities to achieve effective separation between lipid classes, and completes the separation within lipid classes by optimizing chromatographic separation, minimizing the co-elution of lipids, obtaining high-quality secondary mass spectra, and achieving high coverage and accurate qualitative and quantitative analysis of lipids in biological samples. At the same time, lipids are separated within lipid classes according to lipid unsaturation and carbon chain length in the chromatographic column, and separated between lipid classes according to lipid polarity. This retention rule can be used to exclude lipids from false positive ions in the results of lipid identification within and between lipid classes to improve the reliability of lipid identification analysis.

[0069] The present invention proposes that after extracting lipids from biological samples, the lipid types and contents in the samples are first quickly determined by thin layer chromatography; the lipids are then eluted in order of weak polarity, medium polarity and polarity by silica gel chromatography, and the lipids are separated and purified, and then separated by gradient elution of C30, C18 and HILIC columns before entering high-resolution mass spectrometry for detection and analysis. This method can improve the purity of lipids by customizing the silica gel chromatography method, and improve the separation between and within lipid classes by elution separation with liquid chromatography, reduce lipid co-elution behavior, and not only improve the clarity of the secondary mass spectrogram, but also improve the response signal of low-abundance lipids.

[0070] The method provided by the present invention can quickly determine the type and content of lipids contained in the biological sample through thin layer chromatography analysis after biological sample extraction, and can effectively determine the next step of silica gel column separation, thereby reducing solvents and experimental operation procedures;

[0071] The lipids are separated, purified and enriched according to their polarity by using a silica gel chromatographic column, then eluted and separated by liquid chromatography, and then entered into high-resolution mass spectrometry for detection and analysis, which can effectively reduce the co-elution of lipids, increase the content of low-abundance lipids, enhance the mass spectrometry signal response, and improve the coverage rate of lipid detection;

[0072] In the method of the present invention, the retention behavior of lipids in the chromatographic column is stable and the interference within and between lipid classes is small, which is beneficial to judging the false positives of lipid identification through the retention behavior, improving the identification accuracy, reducing the identification difficulty, and saving time cost and economic cost. Description of the Drawings

[0073] Figure 1 It is the TIC diagram of the sample C30 chromatographic column and the lipid retention position diagram in Example 1 of the present application;

[0074] Figure 2 It is the TIC diagram of the sample C18 chromatographic column and the lipid retention position diagram in Example 1 of the present application;

[0075] Figure 3 It is the TIC diagram of the sample HILIC chromatographic column and the lipid retention position diagram in Example 1 of the present application;

[0076] Figure 4 It is the thin-layer chromatogram of the sample lipids in Example 1 and Example 2 of the present application;

[0077] Figure 5 It is the overall comparison diagram of the lipids (major categories) identified in the present application and the reference group in Example 1 of the present application;

[0078] Figure 6 It is the quantity comparison diagram of the lipids (subcategories) identified in the present application and the reference method in Example 1 of the present application;

[0079] Figure 7 It is the m / z vs. RT mapping relationship diagram of TG in the present application and the reference group in Example 1 of the present application;

[0080] Figure 8 It is the m / z vs. RT mapping relationship diagram of DG in the present application and the reference group in Example 1 of the present application;

[0081] Figure 9 It is the chromatogram-mass spectrometry diagram of DG by the method of the present application in Example 1 of the present application;

[0082] Figure 10 It is the chromatogram-mass spectrometry diagram of DG by the reference group method in Example 1 of the present application;

[0083] Figure 11 It is the overall comparison diagram of the lipids (major categories) identified in the present application and the reference group in Example 2 of the present application;

[0084] Figure 12It is a comparison chart of the number of lipids (subclasses) identified by this application and the reference method in Example 2 of this application;

[0085] Figure 13 It is a mapping relationship diagram of M / Z vs RT of this application and reference group PC in Example 2 of this application;

[0086] Figure 14 It is a mass spectrometry acquisition and analysis chart of PC 34:0 in Example 2 of this application;

[0087] Figure 15 It is a mass spectrometry acquisition chart of reference group PC 34:0 in Example 2 of this application;

[0088] Figure 16 It is the solvent selection for the optimal elution efficiency of lipids on the NH2-based SPE column;

[0089] Figure 17 It is the elution recovery rate of lipids on the NH2-based SPE column. Detailed implementation manners

[0090] Abbreviation and definition of key terms

[0091] Liquid chromatography-high resolution mass spectrometry (HPLC-HRMS)

[0092] Lipidomics: A method that can achieve high-throughput detection of lipids

[0093] Column model: C30\C18\HILIC, suitable for the separation of substances with different properties

[0094] High-definition tandem mass spectrometry diagram: The fragment information is consistent with the parent ion attribution, and there are no interfering impurity ions

[0095] Collision cross section (CCS): An inherent property of a substance, which determines the flight time in the drift cell

[0096] Isomers: Having the same molecular weight, generally with similar structures, and it is very difficult to separate them by simple separation. Due to the close retention time and molecular weight, they will enter the mass spectrometry simultaneously, resulting in unclean data acquisition at the mass spectrometry end

[0097] Convolution peak: A chromatographic peak formed by the mixing of multiple ions

[0098] In order to enable those skilled in the art to better understand the solution of this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of this application. The described embodiments are only a part of the embodiments of this application, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts shall fall within the protection scope of this application.

[0099] When numerical ranges are given in the embodiments, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, equipment, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, equipment, and materials of the prior art similar to or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0100] Unless otherwise stated, the experimental methods, detection methods, and preparation methods not disclosed in the present invention all adopt the conventional techniques in the art.

[0101] It should be noted that the term "comprising" in the specification, claims, and above-mentioned drawings of this application is intended to cover non-exclusive inclusion. For example, a process comprising a series of steps does not necessarily have to be limited to those steps clearly listed, but may include other steps not clearly listed or inherent to these processes.

[0102] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. The following will detail this application with reference to the drawings and in combination with the embodiments.

[0103] In the embodiments of this application, after the biological sample tissue homogenate is extracted, thin-layer chromatography analysis is performed to determine the lipid classes and contents contained; a silica gel chromatography column is used to further separate and purify the lipids, and the elution method and steps of the silica gel column are determined in combination with the analysis results of thin-layer chromatography; the separated and purified lipids are separated by liquid chromatography and introduced into a high-resolution mass spectrometer, and software is used to identify and analyze the detected lipids to determine the lipid species information. The method provided in this application improves the separation degree of lipids, especially isomers, reduces the phenomenon of lipid co-elution, obtains high-quality secondary mass spectra, improves the accuracy of lipid qualitative and quantitative analysis, and has the advantages of simple operation, high repeatability, and strong versatility by optimizing the pretreatment of biological sample lipids.

[0104] Example 1 Comparison of Lipid Analysis Results in Zebrafish Brain Tissue

[0105] Extraction method: Both this application and the reference group adopt the modified Folch lipid extraction method. The specific operation process is as follows: Accurately weigh 20 mg of zebrafish brain tissue into a centrifuge tube, add 480 µL of CH 2 Cl 2 :MeOH (2:1 v / v), ultrasonically extract at 4 °C for 10 min (Q800R3 (Qsonica, USA)), add 150 µL of water, centrifuge for 10 min, recover the organic phase, and add 250 µL of CH2 Cl 2 : MEOH (2:1 v / v), repeat once, combine the organic phases, blow-dry with nitrogen, and dissolve in 100 µL CH 2 CL 2 : Dissolve in MeOH (2:1 v / v).

[0106] After the brain lipids of the reference group zebrafish were extracted by the above lipid extraction method, they were passed through a 0.2 μm PTFE filter membrane (Cytiva Pall), then separated by liquid chromatography and analyzed by high-resolution mass spectrometry. The reference group used a CSH C 18 column (Waters 2.1×150 mm, 1.7 µm) for separation, and the column temperature was maintained at 50 °C. Mobile phase A (acetonitrile: water (6:4, v / v)) and mobile phase B (isopropanol: acetonitrile (9:1, v / v)) were used, both containing 10 mM ammonium formate and 0.1% formic acid. The flow rate was 0.28 mL / min, and the elution gradient was as follows: 0, 60% A; 4, 55% A; 4.1, 50% A; 12, 44% A; 12.1, 30% A; 26, 1% A; then re-equilibrated to 60% A in the following 6 min.

[0107] This application is processed according to the chromatographic conditions in item 9 of the present invention. After thin-layer chromatography analysis of the sample, the sample is eluted into three categories of strong, medium, and weak polarities using an NH2 column, and then eluted and analyzed using C30 / C18 / HILIC chromatographic columns respectively. The specific operation steps are as follows:

[0108] 1. Spot the sample on a thin-layer plate (Qingdao Ocean, GF254). ① Develop with n-hexane / ethyl ether (1:1 v / v) to 45% of the height of the silica gel plate. Take out the silica gel plate from the developing tank and wait for the organic solvent to volatilize completely, then develop in the developing agent ② dichloromethane / isopropanol / methanol / formic acid (5:3:2:0.5, volume ratio) to 90% of the height of the silica gel plate, and visualize with iodine. The results are as Figure 4 The thin-layer chromatography shows that the lipids in the sample of Example 1 can be divided into three parts: weak polar lipids, medium polar lipids, and polar lipids.

[0109] Among them, the spotting method is as follows: Use a silica gel thin-layer chromatography plate to spot the sample and the standard on a silica gel plate pre-dried at 60 °C for 5 min: the spotting diameter ≤ 3 mm, 10 mm from the lower edge of the plate, the spotting amount of the standard is 10 μL, and the spotting amount of the sample is 3 μL.

[0110] 2. Select NH 2Base SPE column (Milebio, LBSNH25006), activated with 6 column volumes of n-hexane, and the lipid sample was reconstituted with 1 mL of n-hexane and loaded. Weakly polar lipids were eluted with n-hexane, moderately polar lipids were eluted with isopropanol:dichloromethane (3:7 (v / v)), and polar lipids were eluted with methanol, with an elution volume of 6 column volumes.

[0111] 3. Further, after the eluate was concentrated to dryness, the methanol elution phase was reconstituted with 200 µL of pure methanol, and the other two phases were each reconstituted with 200 µL of dichloromethane:methanol (1:1 (v / v)), and then filtered through a 0.20 µm filter membrane for analysis.

[0112] 4. For liquid chromatography analysis, the n-hexane phase was analyzed using a C30 column, the isopropanol:dichloromethane (3:7 (v / v)) phase was analyzed using a C18 column, and the mobile phases were both A, acetonitrile:water (6:4 (v / v)); B, isopropanol:acetonitrile (9:1 (v / v)), both containing 0.1% formic acid and 10 mM ammonium formate. The methanol phase was analyzed using a HILIC column, and the mobile phases were A, acetonitrile:water (1:3 (v / v)), B, acetonitrile:water (95:5 (v / v)); both A and B contained 0.2% v / v formic acid and 10 mM ammonium formate.

[0113] 5. In this embodiment, the chromatographic conditions are as follows:

[0114] C30 chromatographic column, Thermo Fisher 2.6 µm, 150 x 2.1 mm, elution program: at 0 min, mobile phase B 50% v / v; at 4 min, mobile phase B 70% v / v; at 13 min, mobile phase B 75% v / v; at 24 min, mobile phase B 85% v / v; at 33 min, mobile phase B 90% v / v; at 36 min, mobile phase B 92% v / v; at 37 min, mobile phase B 95% v / v; at 46 min, mobile phase B 95% v / v; at 46.1 min, mobile phase B 50% v / v; at 50 min, mobile phase B 50% v / v; injection volume is 2 µL, column temperature is 50 °C, and flow rate is 0.3 mL / min.

[0115] The C18 chromatographic column is a waters X bridge 3.5μm, 150 x 2.1 mm. Elution program: At 0 min, mobile phase B is 40% v / v; at 3 min, mobile phase B is 50% v / v; at 6 min, mobile phase B is 62% v / v; at 14 min, mobile phase B is 72% v / v; at 15 min, mobile phase B is 85% v / v; at 16 min, mobile phase B is 95% v / v; at 19 min, mobile phase B is 95% v / v; at 19.1 min, mobile phase B is 40% v / v; at 22 min, mobile phase B is 40% v / v. The injection volume is 2 μL, the column temperature is 50 °C, and the flow rate is 0.3 mL / min.

[0116] The Hilic chromatographic column is a waters HILIC 1.7μm, 150 x 2.1 mm. Elution program: At 0 min, mobile phase B is 99% v / v; at 6 min, mobile phase B is 94% v / v; at 11 min, mobile phase B is 92% v / v; at 16 min, mobile phase B is 88% v / v; at 15 min, mobile phase B is 85% v / v; at 16 min, mobile phase B is 95% v / v; at 19 min, mobile phase B is 95% v / v; at 19.1 min, mobile phase B is 40% v / v; at 22 min, mobile phase B is 40% v / v. The injection volume is 2 μL, the column temperature is 40 °C, and the flow rate is 0.3 mL / min.

[0117] Instrumental analysis: Both the reference group and this application use Thermo Scientific Q Exactive for analysis. The mass spectrometry parameters are preferably as follows, and positive and negative modes are used for acquisition; a. The voltages in both positive and negative ion modes are 4.00 kV; b. The ion transfer tube temperature is 320 °C; c. The sheath gas pressures are 40 psi and 30 psi respectively, the auxiliary gas pressure is 10 arb for both, and the auxiliary gas heating temperature is 300 °C for both; d. The scan mode is Full MS, the instrument resolution is set to 70000, -dd / MS2, the resolution is 17500, NCE is 30 eV, and the scan range is 50 - 1200 m / z.

[0118] Data processing: Use MSDIAL to process the data with default parameters. Select the identified lipids according to the highest score, select the lipids with fill% > 50 in the QC samples, with RSD < 30%, and analyze after deleting duplicates.

[0119] Result analysis:

[0120] Figure 1 Shows the TIC chromatogram of sample C30 chromatographic column and the lipid retention position diagram in Example 1 of this application; Figure 2Shows the TIC chromatogram of the sample C18 chromatographic column and the lipid retention position map in Example 1 of the present application; Figure 3 Shows the TIC chromatogram of the sample HILIC chromatographic column and the lipid retention position map in Example 1 of the present application. Figure 4 Shows the thin layer chromatogram of the sample lipid.

[0121] Figure 5 The results showed that in the brain tissue of zebrafish in the reference group, a total of 5 major categories of lipids were detected, including 45 lipid subclasses and 1103 lipid molecules. They are mainly: fatty acids (FA) with a total of 7 subclasses and 65 lipids; glycerolipids (GL) with a total of 8 subclasses and 370 lipids; glycerophospholipids (GP) with a total of 15 subclasses and 387 lipid species; sphingolipids (SP) with a total of 12 subclasses and 275 lipid species; and sterols with 3 subclasses and 6 lipids. After the brain tissue of zebrafish was treated by the method of the present application, 5 major categories of lipids, 52 lipid subclasses, and 1542 lipid species were identified. They are mainly: fatty acids with a total of 9 subclasses and 86 lipids; glycerolipids (GL) with a total of 11 subclasses and 579 lipids; glycerophospholipids (GP) with a total of 15 subclasses and 495 lipids; sphingolipids (SP) with a total of 13 subclasses and 361 lipid species; and sterols with 4 subclasses and 21 lipids.

[0122] Compared with the reference group, the method of the present application not only comprehensively improves the number of lipid identifications but also improves the accuracy of lipid identifications. Figure 6 It shows that the number of lipids identified by the present application is increased by 41.97% compared with the reference group. Among them, glycerolipids are increased by 56.48%; glycerophospholipids are increased by 27.9%; sphingolipids are increased by 31.27%; fatty acids are increased by 32.3%; and sterols are increased by 200%.

[0123] Figure 6 It shows that the number of identifications of glycerolipids TG, DG, and PC in the present application is significantly increased. Among them, the identification rate of TG is 43.06%; DG is increased by 93.75%; and PC is increased by 75%. In order to further verify the accuracy of lipid identification, the present invention maps m / z to RT for the two groups of treatments. Figure 7 It is the mapping relationship diagram of M / Z to RT of TG for the application group and the reference group. The results show that there is a good positive correlation between the carbon number (m / z) and the retention time (RT) of the TG lipid molecules identified by the treatment of the present application. At the same time, as the number of double bonds increases, its retention time decreases, showing a good negative correlation. The results are neat and orderly, in line with the retention behavior of lipid molecules on the chromatogram, proving that the reliability of the lipid identification results is high. The mapping relationship of TG identified in the reference group is relatively complex, indicating that there are interference results such as false positives in the identified lipid molecules, and the reliability is relatively low. The same results are presented in the mapping relationship diagram of m / z and RT of DG, as Figure 8 shown.

[0124] Figure 7 、 Figure 8 The results showed that the same m / z could be detected at multiple retention times, and according to the identification results, they were isomers of TG lipids. Figure 8 The lipid molecule marked in it is DG 38:6, Figure 9 which is the mass spectrometry acquisition and analysis diagram of DG 38:6, Figure 9 In it, A is the chromatographic separation diagram of DG38:6, and it can be seen that there are 3 isomers, Figure 9 In it, B is the corresponding mass spectrometry diagram. The mass spectrometry diagram is clear and the signal response is high. The identification results are: DG 38:6|DG 18:2_20:4, DG 38:6|DG 18:1_20:5, DG 38:6|DG 18:1_20:5.

[0125] Figure 10 which is the mass spectrometry acquisition diagram of the reference group DG 38:6, Figure 10 In it, A is the chromatographic separation diagram. It can be seen that the response intensity decreases and the peak shape is asymmetric, and the separation of the isomers of DG 38:6 is not seen. Figure 10 The mass spectrometry of B in it shows that the identification result is DG38:6|DG 18:2_20:4.

[0126] Example 2 Comparison of lipid analysis results in milk

[0127] The extraction method includes the following steps: Add 1 mL of milk sample to a 15 mL centrifuge tube, and sequentially add 2 mL of methanol (containing 0.2% formic acid by volume) and 4 mL of dichloromethane. After each addition, vortex for 2 min and let it stand in an ice-water bath for 10 min; then add 1 mL of ultrapure water to the centrifuge tube to induce phase separation, vortex for 2 min, and centrifuge at 10000 rpm / min at 4°C for 5 min. Transfer the lower organic phase to a new tube, re-extract the remaining part according to the previous steps, combine the lower organic phases, and dry them under gentle N 2 gas and re-dissolve with 1 mL of dichloromethane:methanol (2:1, v / v). The total lipids obtained are stored at -80°C until use.

[0128] The reference group directly passes through a 0.2 μm PTFE of Whatman and is injected for detection and analysis; the reference group uses Xbridge BEH C 18Separation was performed on a column (Waters 2.1×150 mm, 3.5 µm) maintained at 50 °C. Mobile phase A (acetonitrile: water (6:4, v / v)) and mobile phase B (isopropanol: acetonitrile (9:1, v / v)) were used, both containing 10 mM ammonium formate and 0.1% formic acid. The flow rate was 0.28 mL / min for a total of 32 min, and the elution gradient was as follows: 0, 60% A; 4, 55% A; 4.1, 50% A; 12, 44% A; 12.1, 30% A; 26, 1% A; followed by re-equilibration to 60% A for 6 min.

[0129] The specific operations of this application are as follows:

[0130] 1. After lipid extraction, the sample was spotted on a thin-layer plate (Qingdao Ocean, GF254). ① It was developed with n-hexane / ether (1:1 v / v) to 45% of the height of the silica gel plate. After taking out the silica gel plate from the developing tank and waiting for the organic solvent to volatilize completely, it was then developed in the developing agent ② dichloromethane / isopropanol / methanol / formic acid (5:3:2:0.5, v / v) to 90% of the height of the silica gel plate, and iodine was used for color development. The results are as Figure 4 shown. The lipids in the sample of Example 2 could be divided into weakly polar and polar lipid fractions.

[0131] Among them, the spotting method was as follows: Using a silica gel thin-layer chromatography plate, the sample and the standard were spotted on a silica gel plate pre-dried at 60 °C for 5 min. The spotting diameter was ≤3 mm, 10 mm from the lower edge of the plate. The spotting amount of the standard was 10 μL, and the spotting amount of the sample was 3 μL.

[0132] 2. An NH 2 -based column was selected for separation. It was activated with 6 column volumes of n-hexane, and the lipids were redissolved in 1 mL of n-hexane and loaded. Weakly polar lipids were eluted with n-hexane and polar lipids were eluted with methanol, and the elution volume was 8 column volumes.

[0133] 3. Further, after the eluate was concentrated to dryness, the methanol elution phase was redissolved in pure methanol, and the n-hexane phase was redissolved in dichloromethane: methanol (2:1 (v / v)), and then filtered through a 0.20 µm filter membrane for analysis.

[0134] 4. For liquid chromatography analysis, the n-hexane phase was analyzed using a C30 column. The mobile phases were A, acetonitrile: water (6:4 (v / v)); B, isopropanol: acetonitrile (9:1 (v / v)), and both A and B contained 0.1% formic acid and 10 mM ammonium formate; the methanol phase was analyzed using a HILIC column. The mobile phases were A, acetonitrile: water (1:3 (v / v)), B, acetonitrile: water (95:5 (v / v)). Both A and B contained 0.2% formic acid and 10 mM ammonium formate; the injection volume was 2 μL, the column temperature was 50 °C, and the flow rate was 0.3 mL / min.

[0135] 5. In this embodiment, the chromatographic conditions are as follows:

[0136] C30 chromatographic column Thermo Fisher 2.6 μm, 150 x 2.1 mm. Elution program: at 0 min, mobile phase B 50% v / v; at 4 min, mobile phase B 70% v / v; at 13 min, mobile phase B 75% v / v; at 24 min, mobile phase B 85% v / v; at 33 min, mobile phase B 90% v / v; at 36 min, mobile phase B 92% v / v; at 37 min, mobile phase B 95% v / v; at 46 min, mobile phase B 95% v / v; at 46.1 min, mobile phase B 50% v / v; at 50 min, mobile phase B 50% v / v; injection volume is 2 μL, column temperature is 50 °C, and flow rate is 0.3 mL / min.

[0137] Hilic chromatographic column is waters HILIC 1.7 μm, 150 x 2.1 mm. Elution program: at 0 min, mobile phase B 99% v / v; at 6 min, mobile phase B 94% v / v; at 11 min, mobile phase B 92% v / v; at 16 min, mobile phase B 88% v / v; at 15 min, mobile phase B 85% v / v; at 16 min, mobile phase B 95% v / v; at 19 min, mobile phase B 95% v / v; at 19.1 min, mobile phase B 40% v / v; at 22 min, mobile phase B 40% v / v; injection volume is 2 μL, column temperature is 40 °C, and flow rate is 0.3 mL / min.

[0138] Agilent 6545 instrument analysis: electrospray ionization source, data acquisition in data-dependent mode; primary mass spectrometry acquisition mass range m / z 100 - 1600, secondary mass spectrometry acquisition mass range m / z 50 - 1200; collision energy in positive ion mode is 35 eV; collision energy in negative ion mode is 30 eV; gas temperature is 250 °C, sheath gas temperature is 300 °C; sheath gas flow rate is 12 L / min; capillary voltage in positive ion mode is 3000 V, in negative ion mode is 3000 V; fragmentation voltage is 150 V; cone voltage is 65 V; minimum acquisition rate of MS and MS / MS is 3 spectra / s.

[0139] Data processing: Use MSDIAL to process data with default parameters. Select the identified lipids according to the highest score, select the lipids with fill% > 50 in the QC samples, RSD < 30%, and analyze after removing duplicates.

[0140] Result analysis:

[0141] A total of 31 lipid subclasses and 735 lipid species were detected in milk tissue by software processing of the reference group data. They are mainly: 5 subclasses and 26 lipid species of fatty acids; 6 subclasses and 347 lipid species of glycerolipids (GL); 11 subclasses and 314 lipid species of glycerophospholipids (GP); 7 subclasses and 46 lipid species of sphingolipids (SP); 2 subclasses of sterols and 2 lipid molecules.

[0142] After the treatment by the method of this application and injection for analysis, 38 lipid subclasses and 1062 lipid species were identified. They are mainly: 5 subclasses and 31 lipid species of fatty acids (FA); 7 subclasses and 442 lipid species of glycerolipids (GL); 16 subclasses and 495 lipid species of glycerophospholipids (GP); 8 subclasses and 73 lipid species of sphingolipids (SP); 3 subclasses of sterol lipids and 5 sterol molecules.

[0143] Compared with the reference group, the method of this application comprehensively increases the number of lipid identifications and also improves the accuracy of lipid identification. Figure 11 It shows that the number of lipids identified by this application is increased by 44.48% compared with the reference group. Among them, glycerolipids are increased by 31.98%; glycerophospholipids are increased by 57.64%; sphingolipids are increased by 62.79%; fatty acids are increased by 19.23%; sterols are increased by 150%.

[0144] Figure 12 It shows that the number of identifications of PC, PE, and PC in glycerolipids is significantly increased in this application. The thin-layer results show that the phospholipid content in milk is lower than that of glycerolipids and will be interfered by high-abundance glycerolipids in direct analysis in the reference group. After the treatment of this application, the number of identifications of PC is increased by 69.23%; DG is increased by 93.33%; SM is increased by 81.39%. In order to further verify the accuracy of lipid identification, the present invention maps m / z to RT for the two groups of treatments. Figure 13 It is a mapping relationship diagram of M / Z to RT of PC for the application group and the reference group. The results show that there is a good positive correlation between the carbon number (m / z) and the retention time (RT) of the PC lipid molecules identified by the treatment of this application. At the same time, as the number of double bonds increases, its retention time decreases, showing a good negative correlation. The results are neat and orderly, in line with the retention behavior of lipid molecules on the chromatogram, proving that the reliability of lipid identification results is relatively high. From the mapping relationship of PC identified in the reference group, it can be seen that the lipid detection rate decreases and the retention behavior of lipids is unstable. The results show that there are interferences such as false positives in the signals collected by the mass spectrometry of the reference group compared with the treatment of this application, affecting the accuracy and coverage of lipid identification.

[0145] Figure 14 It is a mass spectrometry acquisition and analysis diagram of PC 34:0. Figure 14In A, it is the chromatogram of PC 34:1. It can be seen that the peak shape is sharp and symmetrical, and the signal intensity is high. Figure 14 In B, it is the mass spectrum of PC 34:0. The mass spectrum is clear and the signal response is high. It is identified as PC 34:0|PC 16:0_18:0. Figure 15 It is the mass spectrum acquisition diagram of the reference group PC 34:0. Figure 15 In A, it is the chromatographic separation diagram with a lower response intensity and an asymmetrical peak shape. Figure 15 In the mass spectrum of B, there are only the parent ion m / z762.5971 and the m / z 184.0751 of the PC head group, and the key information of the acyl chain is missing in the middle. This lipid can only be identified to the subclass, which is PC 34:0.

[0146] According to the comparison of the experimental results of the above embodiments, the lipid analysis method of the present application can quickly determine the lipid category and content in the sample through thin-layer chromatography after lipid extraction; silica gel chromatography separates and purifies lipids according to weak polarity, medium polarity, and polarity, and then gradient custom elution is carried out through C30, C18, and HILIC chromatographic columns, which improves the separation degree between and within lipid classes, reduces the co-elution behavior of lipids, not only improves the clarity of the secondary mass spectrum, but also improves the response signal of low-abundance lipids. Compared with the reference group analysis method, the customization of the present application can increase the lipid molecule concentration and the separation effect of isomers, and improve the coverage rate, reliability, and identification efficiency of lipid detection.

[0147] Comparative example

[0148] Lipids can be classified into three major categories of strongly, moderately, and weakly polar lipids according to thin-layer chromatography. An NH2-based SPE column was selected as the means for further separation and purification. TG (TG 16:0_16:0_16:0, TG 15:0_15:0_15:0, TG 12:0_12:0_12:0, TG10:0_10:0_10:0), DG (DG 20:5_20:5, DG 20:3_20:3, DG 18:3_18:3, DG 15:0_18:1(d7)), SP (HexCer 18:1;O2 / 12:0, HexCer 18:1;O2 / 18:1, Cer 18:1;O2 / 12:0, Cer 18:1;O2 / 18:1, Cer 18:0;O2 / 18:1, SM24:1;O2, SM 36:2;O2, SM 36:1;O2), PC (PC 16:0_18:1, PC 15:0_18:1(d7), PC 14:0_14:0), PE (PE 16:0_18:1, PE 15:0_18:1(d7), PE 15:0_15:0), MG 18:0, LPC 18:1(d7), C18(plasm)-18:1(d9) PC standards were used. The sample was loaded with n-hexane (HEX), and then successively eluted with HEX, HEX:DCM 7:3, HEX:DCM 5:5, HEX:DCM 3:7, DCM, DCM:IPA 7:3, DCM:IPA 5:5, DCM:IPA3:7, IPA, IPA:MeOH 7:3, IPA:MeOH 5:5, IPA:MeOH 3:7, MeOH, MeOH+FA1% (volume ratio). The elution volume was 3 times the column volume (3 times the column volume is 3 mL, Figure 16 and the 6 mL of HEX in

[0149] was eluted with 3 times the column volume twice), and then analyzed by mass spectrometry. Figure 16 As shown in

[0150] After selecting strong, medium, and weak polar elution solvents for lipids, the elution solvents and efficiency were optimized by adding Avanti lipid internal standards LM6004, LM6002, LM6000, and 330709w. The elution recovery results showed that TG lipid LM6000 was mainly eluted in the HEX phase, SP lipid LM6002 (except SM), and DG lipid LM6004 were mainly eluted in DCM:IPA 7:3, and the internal standard 330709w GP lipid and SM lipid had the best elution effect in the methanol phase (as Figure 17 shown). The elution volume was 6 times the column volume.

[0151] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for analyzing lipids in a biological sample, characterized in that: The method for analyzing lipids in a biological sample comprises the following steps: (1) extracting lipids from a biological sample to obtain a first sample; (2) determining the lipid categories of the first sample by thin layer chromatography, wherein the lipid categories include non-polar lipids, medium-polar lipids, and polar lipids; (3) according to the lipid types in (2), separating and purifying the first sample using a silica gel chromatography column to obtain a second sample; (4) performing gradient elution on the second sample using liquid chromatography and then performing mass spectrometry analysis; The silica gel chromatographic column described in step (3) is an NH2-based SPE column; the eluent of the silica gel chromatographic column includes a n-hexane phase, an isopropanol:dichloromethane phase with a volume ratio of 3:7 and a methanol phase, or a n-hexane phase and a methanol phase; the n-hexane phase is used to elute non-polar lipids; the isopropanol:dichloromethane phase with a volume ratio of 3:7 is used to elute medium-polar lipids; the methanol phase is used to elute polar lipids; In step (4), the C30 column is used to separate and purify the sample eluted with the n-hexane phase, the C18 column is used to separate and purify the sample eluted with the isopropanol:dichloromethane phase in a volume ratio of 3:7, and the HILIC column is used to separate and purify the sample eluted with the methanol phase.

2. The method for analyzing lipids in a biological sample according to claim 1, characterized in that: The mobile phase system of the C30 chromatographic column or C18 chromatographic column is: A1: acetonitrile: water in a volume ratio of 6:4; B1: isopropanol:acetonitrile with a volume ratio of 9:1, 5-10 mM ammonium formate and 0.1%-0.2% formic acid were added to both A1 and B1; The mobile phase system of the HILIC chromatographic column is: A2: acetonitrile: water in a volume ratio of 1:3; B2: acetonitrile: water in a volume ratio of 95:

5. 5-10 mM ammonium formate and 0.1%-0.2% formic acid were added to both A2 and B2.

3. The method for analyzing lipids in a biological sample according to claim 1, characterized in that: The liquid chromatography elution procedure in step (4) is: Elution program of C30 column: at 0 min, mobile phase B1 50% v / v; at 4 min, mobile phase B1 70% v / v; at 13 min, mobile phase B1 75% v / v; at 24 min, mobile phase B1 85% v / v; at 33 min, mobile phase B1 90% v / v; at 36 min, mobile phase B1 92% v / v; at 37 min, mobile phase B1 95% v / v; at 46 min, mobile phase B1 95% v / v; at 46.1 min, mobile phase B1 50% v / v; at 50 min, mobile phase B1 50% v / v; C18 column elution program: at 0 min, mobile phase B1 40% v / v; at 3 min, mobile phase B1 50% v / v; at 6 min, mobile phase B1 62% v / v; at 14 min, mobile phase B1 72% v / v; at 15 min, mobile phase B1 85% v / v; at 16 min, mobile phase B1 95% v / v; at 19 min, mobile phase B1 95% v / v; at 19.1 min, mobile phase B1 40% v / v; at 22 min, mobile phase B1 40% v / v; HILIC column elution program: at 0 min, mobile phase B2 99% v / v; at 6 min, mobile phase B2 94% v / v; at 11 min, mobile phase B2 92% v / v; at 16 min, mobile phase B2 88% v / v; at 15 min, mobile phase B2 85% v / v; at 16 min, mobile phase B2 95% v / v; at 19 min, mobile phase B2 95% v / v; at 19.1 min, mobile phase B2 40% v / v; at 22 min, mobile phase B2 40% v / v.

4. The method for analyzing lipids in a biological sample according to claim 3, characterized in that: In step (4), the mass spectrum is collected in CID mode to obtain the accurate ion mass-to-charge ratio, retention time and high-definition secondary mass spectrum of the lipid primary mass spectrum; in step (4), the mass spectrum is collected in the following ways: using positive ion mode to detect and analyze the effluent after separation and purification by C30 chromatographic column; using positive and negative modes to detect and analyze the effluent after separation and purification by C18 chromatographic column; using positive and negative modes to detect and analyze the effluent after separation and purification by HILIC chromatographic column.

5. The method for analyzing lipids in a biological sample according to claim 3, characterized in that: The elution conditions of the chromatographic column are: the flow rate of the mobile phase is 0.2-0.4 mL / min, and the column temperature is 35-50°C.

6. The method for analyzing lipids in a biological sample according to any one of claims 1 to 5, characterized in that: The biological sample includes a sample containing lipids or lipid analogs.

7. The method for analyzing lipids in a biological sample according to any one of claims 1 to 5, characterized in that: The method for extracting lipids from the biological sample in step (1) is: Add an extractant to the biological sample, ultrasonically disrupt or let it stand, then add water and centrifuge, collect the organic phase, then repeat the extraction of the remaining part, combine the organic phases, remove the solvent, redissolve, filter, and obtain the first sample; the extractant includes methanol and dichloromethane.

8. The method for analyzing lipids in a biological sample according to any one of claims 1 to 5, characterized in that: Step (2) includes: Spot the first sample and the standard using a silica gel thin layer chromatography plate; The samples were developed by the secondary development method. The developing agents were ① n-hexane / ethyl ether, ② dichloromethane / isopropanol / methanol / water / formic acid. After development, the samples were taken out and the color was developed in a color developing tank after the reagents were completely evaporated.

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